NGR-based peptides as molecular imaging agents of neovascularization after myocardial infarction

Objectives: Angiogenesis plays an important role in restoration of blood perfusion after myocardial infarction (MI). The receptor CD13 is selectively expressed on angiogenic endothelium and binds the tripeptide motif Asn-Gly-Arg (NGR). Aim of this study was to design and synthesize cyclic Asn-Gly-Arg (NGR)-based imaging probes with improved stability and efficacy for non-invasive SPECT imaging of angiogenesis.

Methods: Linear CNGRG-MpaL thioester peptide was synthesized by manual Boc-based solid-phase peptide synthesis and cyclized using native chemical ligation instead of disulfide bridging. This monomeric cyclic backbone coNGR peptide contained a sulfhydryl group that was conjugated to maleimide-DTPA. Additional to this monomer, a tetrameric coNGR peptide was synthesized by coupling coNGR to a tetrameric scaffold containing a thiaproline (Thz) at the core. The sulfhydryl group of Thz was decrypted and reacted with maleimide-DTPA. Resulting DTPA-[SMCC-coNGR]₄ and its monomeric counterpart DTPA-coNGR were radiolabeled with ¹¹¹InCl₃. Angiogenesis after MI was visualized in combination with ^99mTc-sestamibi in dual-isotope micro-SPECT imaging in a mouse model of MI. CD13 immunohistochemistry was performed to validate co-localization of coNGR peptide uptake with CD13 expression. Blood stability was examined using HPLC and biodistribution patterns were evaluated.

Results: In vitro studies indicated better stability for both novel imaging agents after 50 min blood incubation compared to the previously reported disulfide-cyclized cNGR imaging agent. Uptake patterns of ¹¹¹In-labeled mono- and tetrameric coNGR peptides coincided with CD13 immunohistochemistry on excised hearts. In addition, tetrameric coNGR showed a significantly higher specific uptake in infarcted myocardium compared to monomeric coNGR imaging agent. Dual-isotope SPECT allowed simultaneous imaging of CD13-positive angiogenic endothelium and perfusion in the border zone of infarcted myocardium.

Conclusion: Backbone-cyclized angiogenesis SPECT agents, mono- and tetrameric coNGR demonstrated a markedly higher stability in blood compared to disulfide-cyclized cNGR. Radiolabeled tetrameric coNGR is a promising sensitive imaging agent for detection of angiogenesis in infarcted myocardium. Research Support: This study was performed within the framework of the Center for Translational Molecular Medicine, project EMINENCE (grant 01C-204 to GH; TH; MP; ID), the Weijerhorst Foundation (MB; FM) and The Netherlands Organisation for Scientific Research (NWO; VIDI 723.013.009 to ID).